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Current laboratory diagnosis of Q fever
Current Laboratory Diagnosis of Q Fever Bernard La Scola, MD, PhD Q fever is a worldwide zoonosis caused by the strictly intracellular bacterium Coxiella burnetii. Among symptomatic patients (one-half of patients remain asymptomatic), acute Q fever most frequently manifests as a self-limited febrile illness, pneumonia, or hepatitis. Endocarditis is the predominant form of chronic Q fever. All the classical techniques of bacteriology may be used for diagnosis of C burnetii infection. Nonetheless, because of the risk of contamination, isolation must be performed in biosafety level 3 laboratories. Moreover, to date no diagnostic tests for detection by polymerase chain reaction or specific antibodies for immunochemistry are available commercially. Hence, Q fever is diagnosed in most cases by serology. The most reliable technique appears to be micro-immunofluorescence, which exhibits both good sensitivity and specificity. A wider use of this serology in cases of blood culture–negative endocarditis, atypical pneumonia, unexplained fever, and hepatitis should lead to an increase of diagnosed cases. Copyright 2002, Elsevier Science (USA). All rights reserved.
(for query) fever is a worldwide zoonosis. The disease was described first in 1937 by Edward Holbrook Derrick (1898-1976), a public health official in Queensland, Australia, while investigating an outbreak of an unknown febrile illness among abattoir workers. Sir Frank McFarlane Burnet and Mavis Freeman isolated a gram-negative intracellular bacterium from a mouse infected with material sent from Derrick. At the same time, Davis and Herald Rae Cox at the Rocky Mountain Laboratory in Montana isolated an organism from ticks collected near Nine Mile Creek. In 1938, researchers found that the Nine Mile agent and the Q fever agent were identical. The agent was named Coxiella burnetii to honor Cox and Burnet. Since then, C burnetii has been isolated from several animal species. Farm animals and pets are the main reservoirs of infection for humans. Humans acquire the infection mainly via inhalation of infected aerosol particles or ingestion of unpasteurized dairy products. Q fever manifests with a wide spectrum of clinical syndromes. After acquiring primary infection with C burnetii, one-half of patients remain asymptomatic. Among those who are symptomatic, acute Q fever most frequently manifests as a self-limited febrile illness, pneumonia, or hepatitis. Endocarditis is the predominant
Q
From the Unite´ des Rickettsies, CNRS UMR 6020, Faculte´ de Me´decine de Marseille, Marseille, France. Address correspondence to Bernard La Scola, MD, PhD, Unite´ des Rickettsies, CNRS UMR 6020, Faculte´ de Me´decine de Marseille, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France; e-mail: [email protected] Copyright 2002, Elsevier Science (USA). All rights reserved. 1045-1870/02/1304-0006$35.00/0 doi:10.1053/spid.2002.127199
form of chronic Q fever. Despite the availability of wellestablished epidemiologic associations and reliable serologic tests, Q fever infrequently is diagnosed in the clinical setting.1 The diagnosis of Q fever relies mainly upon serology, the method most commonly used being the immunofluorescence assay. This serologic testing always should be done in a patient with a febrile illness and blood cultures that yield negative results.
Q Fever The Bacterium Coxiella burnetii is a small, obligate intracellular, gram-negative bacterium. It is the single species of the genus Coxiella, which belongs to the gamma subdivision of Proteobacteria. Based on 16S rRNA sequence analysis, C burnetii is phylogenetically closest to Legionella spp. C burnetii demonstrates marked resistance to physicochemical agents because of its spore-like form. It resists a temperature of 60°C for 60 minutes and exposure to 0.5 percent formalin for 4 days, and it can survive in soil and milk for several months. In cell cultures or embryonated eggs, C burnetii exhibits phase variation linked to a chromosomal deletion that leads to a lipopolysaccharide shift, with the highly virulent phase I form shifting to the avirulent phase II form. Isolates from different areas of the world exhibit a low degree of genetic heterogeneity.1,2
Epidemiology Q fever has been described in every country except New Zealand. Most cases have been reported from Europe, mainly France, England, Switzerland, Germany, Spain, Greece, Bulgaria, and Israel. Nova Scotia, Canada accounts
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for most of the cases reported from North America. Farm animals, namely cattle, sheep, and goats, are the main reservoirs of C burnetii infection for humans. High numbers of the bacterium are found in the placenta of infected parturient animals and are shed into the environment after labor or abortion. Transmission of infection to humans is accomplished mainly via inhalation of contaminated aerosols generated in this way. Spread of infected aerosols by wind has been reported and may explain cases with no apparent contact with sources of infection.1 In Europe, most cases of Q fever occur during the first 6 months of the year as a result of the heavily contaminated environment after lambing season. Occasional contact with only a small number of bacteria may be sufficient to cause illness.1,3 Outbreaks of Q fever after contact with the birth products of cats, dogs, and other pets also have been reported, mainly from North America, and show no seasonality.4,5 C burnetii also has been isolated from birds and domestic poultry, and humans4,5 may be infected via inhalation of infected fomites.6 The incubation period of acute Q fever ranges from 1 to 3 weeks among patients infected via the respiratory route and depends on the infectious burden.1 Ingestion of unpasteurized dairy products constitutes another source of infection with C burnetii. Cases of human Q fever acquired after contact with parturient products of an infected woman (in an obstetrician), through transplacental transmission during human pregnancy, and through blood and sexual transmission, also have been reported.1
Clinical Manifestations The incidence of Q fever pneumonia is difficult to estimate because of the absence of systematic investigations for infections of C burnetii worldwide. In Nova Scotia, Canada, an active surveillance of the role of Q fever in communityacquired pneumonia is ongoing. Among 149 patients with atypical pneumonia, C burnetii was the third most frequently identified agent (2.7% of the cases), after Mycoplasma pneumoniae and Chlamydia pneumoniae (22.8% and 10.7%, respectively).1 Q fever pneumonia in children has been reported rarely in the literature and most probably remains underdiagnosed.7,8 Patients with Q fever pneumonia present with high-grade fever, in association with severe headache and constitutional symptoms, such as fatigue and myalgia. The incidence of cough ranges from 24 to 90 percent and is productive in one-third of them. Respiratory distress and thoracic pain also are encountered frequently. On auscultation, inspiratory crackles predominate. The most common chest radiographic patterns are single or multiple, frequently bilateral infiltrates, and reticular markings, with a predilection for the lower lobes. Atelectasis and pleural effusion also are described frequently. The course of Q fever pneumonia usually is mild to moderate, compatible with “atypical pneumonia.” However, rapid progression to respiratory failure has been reported.1,7-9 A fatal Q fever pneumonia in an 11-year-old boy with chronic granulomatous disease has been described.10 No specific treatment against Q fever was administered in that case. C burnetii was identified in autopsy lung tissue.
Pathogenesis and Immunity After primary infection has occurred, C burnetii enters monocytes and macrophages via attachment to integrins. Depending on the route of infection (respiratory or gastrointestinal), alveolar macrophages in the lungs or Kupffer cells in the liver are the primary target cells. The avirulent phase II form is destroyed rapidly via the phagolysosomal pathway, whereas the virulent phase I form remains viable. Within macrophages, C burnetii exists in large acidic vacuoles that promote its metabolism and multiplication.1,2 During acute infection, IgM antibodies against phase I and II C burnetii antigens and IgG antibodies against phase II antigens develop. In patients with chronic Q fever, high levels of IgG and IgA antibodies against phase I and II are present. Control of C burnetii infection depends on T cell– mediated immune mechanisms. However, complete eradication of bacteria is not achieved. Patients with malignancies, those infected with human immunodeficiency virus (HIV), and pregnant women may experience relapse of infection, after having an initial response, that may become chronic and frequently fatal. Dysregulation of cytokine responses appears to explain the impaired immunity in such patients. Biopsy specimens taken during acute infection with C burnetii reveal a profound cellular response with a few organisms. However, during chronic infection, biopsy specimens demonstrate high numbers of organisms but a limited cellular response.1,2
Laboratory Diagnosis Nonspecific Laboratory Diagnosis In patients with acute Q fever,11 the leukocyte count usually is normal. However, 25 percent of patients have an elevated white blood cell count (WBC) that ranges from 14 to 21 ⫻ 109/L. The erythrocyte sedimentation rate may be elevated. Thrombocytopenia is noted in 25 percent of cases. Liver enzymes are elevated in as many as 85 percent of cases. The increase in transaminase rate usually is moderate. During an episode of prolonged fever, the association of a normal WBC count, thrombocytopenia, and elevated hepatic enzymes are diagnostic signs of Q fever. In Q fever meningoencephalitis, a mild lymphocytic pleiocytosis frequently is noted in the spinal fluid. Autoantibodies are found frequently during the course of Q fever, but their real significance remains unknown.12-15 In chronic Q fever, clinical and biological symptoms are related to the predominantly cell-mediated inflammatory response to the microorganism.11,16,17 Conventional blood cultures remain negative. The laboratory manifestations of an inflammatory syndrome are the usual ones and include anemia, elevated erythrocyte sedimentation rate, and polyclonal hypergammaglobulinemia. The WBC count may be normal, elevated, or decreased. Thrombocytopenia and elevated hepatic enzyme levels are found commonly. Renal involvement is a common event and is characterized by elevated creatinine levels and microhematuria. Monoclonal immunoglobulins rarely are observed,18 whereas cryoglobulins are found fre-
Current Laboratory Diagnosis of Q Fever quently. Autoantibodies also are frequent findings in chronic Q fever.12,13
Specific Laboratory Diagnosis Samples. C burnetii is a very infectious disease. Thus, only biosafety level 3 laboratories and experienced personnel should be allowed to manipulate contaminated specimens and cultivate this microorganism from clinical samples. Several human specimens are suitable for the detection of C burnetii, but their availability depends on the clinical presentation. DNA amplification may be performed on blood, cerebrospinal fluid, bone marrow, cardiac valve biopsy, vascular aneurysm or graft, bone biopsy, liver biopsy, milk, placenta, fetal specimens in case of an abortion, and cell culture supernatants. Blood should be collected on EDTA or sodium citrate, and the leukocyte layer should be saved for gene amplification. Solid specimens should be kept frozen at -80°C before testing. Cultivation of C burnetii may be obtained from the buffy coat of heparinized blood, whole blood, plasma, bone marrow, cerebrospinal fluid, cardiac valve biopsy, vascular aneurysm or graft, bone biopsy, liver biopsy, milk, placenta, and fetal specimens in case of an abortion, but not from blood collected on EDTA or sodium citrate. All specimens, excluding whole blood, should be stored at -80°C and be forwarded on dry ice to the diagnostic laboratory. Whole blood should be kept at 4°C. Furthermore, red blood cells should not be inoculated onto shell vials because they lead to a high background on examination with ultraviolet light. Pathology, Immunohistochemistry. The immune response during Q fever is associated with an inflammatory reaction that results in formation of granulomatous lesions most commonly involving the lungs, liver, and bone marrow. The histology of Q fever pneumonia in humans rarely has been studied.19-22 Interstitial edema and infiltration by lymphocytes and macrophages occur. Alveolar spaces are filled with histiocytes, and intra-alveolar focal necrosis and hemorrhage have been described. A necrotizing bronchitis and bronchiolitis may be encountered. Microorganisms usually are lacking in these lesions. The hepatic lesions are different in acute and chronic Q fever. In acute cases, the characteristic findings are granulomatous lesions containing the so-called doughnut granuloma, which consists of dense fibrin rings surrounding a central lipid vacuole.23-27 Granulomatous changes and necrosis have been noted in bone marrow.27-29 In chronic cases, pathologic findings are nonspecific with lymphocytic infiltration and foci of spotty necrosis.19 The vegetations in Q fever endocarditis often are smooth and nodular. The valve often is infiltrated with foamy macrophages that are filled with C burnetii cells. The detection of C burnetii in tissues can be achieved on fresh or formalin-fixed and paraffin-embedded samples. Valvular or vascular samples are most valuable. Several techniques are available and include immunoperoxidase,30 a capture system [enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunofluorescent assay (ELIFA)31], or monoclonal antibodies.31-33 The last technique also can be employed for antigenic detection in paraffin-embedded tissues.34
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DNA Detection. The polymerase chain reaction (PCR) has been used successfully to detect C burnetii DNA in cell cultures and clinical samples.35 The availability of primers derived from genes specific to C burnetii has allowed a simple and reliable method for the detection of this bacterium, even in paraffin-embedded tissues.35,36 Moreover, PCR has proven to be more sensitive for retrospective diagnosis on frozen samples and for follow-up of patients treated for chronic Q fever than are standard culture techniques.35 In our experience, specimens kept frozen at -80°C are suitable for PCR for several years. In our laboratory, we routinely use primers derived from the htpAB-associated repetitive element.37,38 This element exists in at least 19 copies in the C burnetii Nine Mile I genome, and PCR based upon this gene is very sensitive.38 Culture. The isolation of C burnetii must be carried out only in biosafety level 3 laboratories because of its extreme infectivity. This microorganism can be isolated by inoculating specimens onto conventional cell cultures (monkey kidney cells—Vero cells), into embryonated hen yolk sacs,39 or into laboratory animals, such as mice or guinea pigs.40 Embryonated eggs die 7 to 9 days after inoculation. Guinea pigs develop fever 5 to 8 days after intraperitoneal inoculation. The spleen is the most valuable organ for the recovery of C burnetii. Ground spleen extracts should be inoculated subsequently into embryonated eggs. The recent development of a cell microculture system from a commercially available method for virus culture, the shell vial cell culture system, has allowed for improvement in the isolation of intracellular bacteria, especially C burnetii.41,42 Specimens are inoculated onto human embryonic lung fibroblasts grown on a 1-cm2 coverslip within a shell vial. A 1-hour centrifugation step enhances attachment and penetration of bacteria into cells. After an incubation period of 6 days, detection of C burnetii within the cells is achieved by microscopic examination after staining. The organism appears as short rods that are not Gram-stained but are visible after Giemsa or Gimenez staining.43 The identification of C burnetii within the cells is performed with a direct immunofluorescence assay using polyclonal or monoclonal anti–C burnetii antibodies conjugated to fluorescein isothiocyanate.42 The detection by antibodies may be confirmed by PCR performed on the shell vial supernatants. Best results for culture are obtained if clinical specimens are collected before initiating antibiotic therapy.44 Fifteen percent of untreated patients with Q fever pneumonia have blood cultures that test positive by this method, as do 53 percent of endocarditis cases.44,45 Serologic diagnosis. Because arriving at the clinical diagnosis is difficult, in most instances, the diagnosis of Q fever relies upon serology. Several methods have been described: microagglutination,46-48 complement fixation,49-51 radioimmunoassay,52 indirect immunofluorescence antibody tests (immunofluorescence assay),51,53 indirect hemolysis test,54 ELISA,55-59 ELIFA,60 dot immunoblotting, and Western immunoblotting.61,62 Criteria to be taken into account in choosing a diagnostic test include its specificity, sensitivity, positive predictive value, cost, and the amount of antigen required. The most reliable and commonly used
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methods are indirect immunofluorescence, complement fixation, ELISA, and microagglutination. Only the first 2 are commercially available and will be described in this article. Currently, the immunofluorescence assay is the reference method for the serodiagnosis of Q fever.63 It is the simplest and one of the most accurate serologic techniques. To prepare antigens for this test, phase II C burnetii Nine Mile reference strain is grown in confluent layers of L929 mouse fibroblasts, and phase I antigens are obtained from the mice spleens inoculated with phase II organisms.64 This method of preparation has been demonstrated to yield antigens with the highest sensitivity for C burnetii antibody detection.65 In our laboratory, we use a micro-immunofluorescence technique, which requires very small amounts of antigens. Sera are diluted in phosphate buffered saline with 3 percent nonfat powdered milk to avoid nonspecific fixation of antibodies. This method can be used to determine antibodies to phases I and II in the IgG, IgM, and IgA fractions. However, test results can be confounded by the presence of a rheumatoid factor. Thus, a rheumatoid factor absorbant is used to remove IgG before the determination of IgM and IgA.64 The choice of a negative cutoff titer depends upon the source and purity of the antigen and the amount of background antigen stimulation in the population to be studied. We use a 1:50 dilution as our first positive dilution.64 Screening is performed with anti–phase II anti-immunoglobulins with a 1:50 dilution for the tested sera. Positive sera then are diluted serially and tested for the presence of anti–phase I and II IgG, IgM, and IgA. Seroconversion usually is detected 7 to 15 days after the onset of clinical symptoms. Approximately 90 percent of patients have detectable antibodies by the third week. Complement fixation is very specific, although less so than is immunofluorescence assay, but it lacks sensitivity.51 Sera are heat-inactivated before testing against phase II antigens.49 This method detects both anti–phase I and II antibodies. However, a prozone phenomenon may be present with serum specimens from patients with chronic Q fever that could result in a false-negative test. Complement fixation also is more time-consuming than is immunofluorescence assay.66 Moreover, cross-reaction with hen egg antigens may result in false-positive results. The interpretation of results requires acute and convalescent phase serum samples. Seroconversion is detected later with the complement fixation test than with immunofluorescence assay or ELISA (between 10 and 20 days after the onset of symptoms).51,67 The predictive value of positive and negative results is affected by the prevalence of the disease, which creates a certain degree of background antigen stimulation in the studied population.68 Therefore, the determination of cutoff values is very important for the interpretation of the serologic results. For example, we use a 1:50 serum dilution for immunofluorescence assay screening, whereas Marrie and Pollak69 used a 1:8 dilution. Whereas a diagnostic test must be very sensitive, a seroepidemiology test must be very specific to prevent false-positive results caused by cross-reacting antibodies. The antigenic variation of C burnetii is extremely useful in differentiating acute and
chronic illness. In acute Q fever, antibodies to phase II antigens predominate, and their titer is higher than the phase I antibody titer. As with many other infectious diseases, IgM antibodies are the first to appear. On the other hand, in chronic forms of the disease, such as endocarditis, elevated anti–phase I antibodies are detected uniformly. For cutoff values in the immunofluorescence assay test, Tissot-Dupont et al64 recommend titers of anti–phase II IgG of 200 or greater and titers of anti–phase II IgM of 50 or greater for the diagnosis of acute Q fever, and titers of anti–phase I IgG of 800 or greater for the diagnosis of chronic Q fever. They also have demonstrated that anti– phase I IgA titers did not contribute to the diagnosis of chronic Q fever. Recently, we demonstrated that an anti– phase I IgG C burnetii titer of 1:800 or greater was diagnostic of Q fever in a case of endocarditis, and, thus, we have modified the Duke criteria for the diagnosis of Q fever endocarditis to include 1 positive blood culture for C burnetii and a phase I IgG titer of 1:800 or greater as major criteria for the diagnosis of Q fever endocarditis.70 A complement fixation titer of 1:40 is diagnostic for acute Q fever,67 and a 1:200 titer of antibody to phase I is diagnostic for chronic Q fever.66 In acute Q fever, immunofluorescence assay titers reach their maximum levels 4 to 8 weeks after the onset of disease and then decrease gradually during the ensuing 12 months.67 Dupuis et al71 have shown that IgM titers declined to undetectable levels after 10 to 12 weeks, whereas the decline took 17 weeks in the study of Field et al.53 Complement fixation and immunofluorescence assay can be combined for the follow-up. A drop in complement fixation titer often implies resolution and will occur before an immunofluorescence assay drop. The persistence of high levels of anti–phase I antibodies despite appropriate treatment, or the reappearance of such antibodies, should raise suspicion of possible chronic Q fever. Patients with valvular or vascular abnormalities, those who are immunodeficient, and pregnant women should have repeated C burnetii serology tests if they have a medical history of acute Q fever or a prolonged and unexplained febrile episode. In the case of an acute Q fever in such a patient, an immunofluorescence assay test should be done monthly for at least 6 months. The follow-up of patients treated for chronic Q fever also should be done serologically. During therapy, serologic testing should be performed once monthly for 6 months and every 3 months thereafter. The levels of antibodies decrease very slowly. When present, IgM antibodies disappear first, then IgA antibodies, but IgG titers remain positive for years. Antimicrobial treatment can be stopped after 18 months to 3 years if the anti–phase I IgG titer by immunofluorescence assay is below 1:400 and anti–phase I IgA is undetectable.14,72
Conclusion Q fever remains a poorly understood disease despite recent advances in the knowledge of its immunopathology. Acute cases present most often as asymptomatic infections, in-
Current Laboratory Diagnosis of Q Fever cluding flulike syndrome, pneumonia, or hepatitis. Host factors probably play an important role in the development of chronic disease, which presents in most cases as a blood culture–negative endocarditis. Although its exact prevalence is unknown, the number of cases of Q fever most likely is underestimated. Therefore, such a diagnosis must be considered in cases of unexplained fever, especially if the fever occurred after contact with possibly contaminated mammals. The best tests for diagnosis are those that permit the direct detection of bacteria. They include the shell vial cell culture, PCR, and the immunodetection on tissue biopsies. All these techniques require a level 3 biosafety laboratory and trained personnel because of the extreme infectivity of C burnetii. In chronic cases, the techniques that allow direct detection of C burnetii in blood or tissues should be used before the beginning of therapy. As for indirect specific diagnosis, the technique to be used should be very sensitive and should detect antibodies early in the course of the disease. Although many techniques have been described, immunofluorescence assay is the reference method. It is both very specific and sensitive. In the case of acute Q fever, diagnosis can be confirmed by an immunofluorescence assay titer greater than the cutoff value (which has to be determined for each area) or by a 4-fold increase in the antibody titer detected by immunofluorescence assay, complement fixation, ELISA, or microagglutination.
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39. Ormsbee RA. The growth of Coxiella burnetii in embryonated eggs. J Bacteriol 63:73-86, 1952 40. Williams JC, Thomas LA, Peacock MG: Humoral immune response to Q fever: Enzyme-linked immunosorbent assay antibody response to Coxiella burnetii in experimentally infected guinea pigs. J Clin Microbiol 24:935-939, 1986 41. Marrero M, Raoult D: Centrifugation-shell vial technique for rapid detection of Mediterranean spotted fever rickettsia in blood culture. Am J Trop Med Hyg 40:197-199, 1989 42. Raoult D, Vestris G, Enea M: Isolation of 16 strains of Coxiella burnetii from patients by using a sensitive centrifugation cell culture system and establishment of strains in HEL cells. J Clin Microbiol 28:2482-2484, 1990 43. Gimenez DF: Staining rickettsiae in yolk-sac cultures. Stain Technol 39:135-140, 1964 44. Musso D, Raoult D: Coxiella burnetii blood cultures from acute and chronic Q-fever patients. J Clin Microbiol 33:3129-3132, 1995 45. Gil-Grande R, Aguado JM, Pastor C, et al: Conventional viral cultures and shell vial assay for diagnosis of apparently culturenegative Coxiella burnetii endocarditis. Eur J Clin Microbiol Infect Dis 14:64-67, 1995 46. Fiset, P, Ormsbee RA, Silberman R, et al: A microagglutination technique for detection and measurement of rickettsial antibodies. Acta Virol 13:60-66, 1969 47. Kazar J, Brezina R, Schramek S, et al: Suitability of the microagglutination test for detection of post-infection and postvaccination Q fever antibodies in human sera. Acta Virol 25: 235-240, 1981 48. Nguyen SV, Otsuka H, Zhang GQ, et al: Rapid method for detection of Coxiella burnetii antibodies using high-density particle agglutination. J Clin Microbiol 34:2947-2951, 1996 49. Herr S, Huchzermeyer HF, Te Brugge LA, et al: The use of a single complement fixation test technique in bovine brucellosis, Johne’s disease, dourine, equine piroplasmosis and Q fever serology. Onderstepoort J Vet Res 52:279-282, 1985 50. Murphy AM, Field PR: The persistence of complement-fixing antibodies to Q-fever (Coxiella burnetii) after infection. Med J Aust 1:1148-1150, 1970 51. Peter O, Dupuis G, Burgdorfer W, et al: Evaluation of the complement fixation and indirect immunofluorescence tests in the early diagnosis of primary Q fever. Eur J Clin Microbiol Infect Dis 4:394-396, 1985 52. Doller G, Doller PC, Gerth HJ: Early diagnosis of Q fever: Detection of immunoglobulin M by radioimmunoassay and enzyme immunoassay. Eur J Clin Microbiol Infect Dis 3:550553, 1984 53. Field PR, Hunt JG, Murphy AM: Detection and persistence of specific IgM antibody to Coxiella burnetii by enzyme-linked immunosorbent assay: A comparison with immunofluorescence and complement fixation tests. J Infect Dis 148:477-487, 1983 54. Tokarevich NK, Schramek S, Daiter AB: Indirect haemolysis test in Q fever. Acta Virol 34:358-360, 1990 55. Kovacova E, Gallo J, Schramek S, et al: Coxiella burnetii antigens for detection of Q fever antibodies by ELISA in human sera. Acta Virol 31:254-259, 1987
56. Peter O, Dupuis G, Bee D, et al: Enzyme-linked immunosorbent assay for diagnosis of chronic Q fever. J Clin Microbiol 26:1978-1982, 1988 57. Uhaa IJ, Fishbein DB, Olson JG, et al : Evaluation of specificity of indirect enzyme-linked immunosorbent assay for diagnosis of human Q fever. J Clin Microbiol 32:1560-1565, 1994 58. Waag D, Chulay J, Marrie TJ, et al: Validation of an enzyme immunoassay for serodiagnosis of acute Q fever. Eur J Clin Microbiol Infect Dis 14:421-427, 1995 59. Williams JC, Thomas LA, Peacock MG: Identification of phasespecific antigenic fractions of Coxiella burnetti by enzyme-linked immunosorbent assay. J Clin Microbiol 24:929-934, 1986 (published erratum in J Clin Microbiol 25:588, 1987) 60. Schmeer N, Muller HP, Baumgartner W, et al: Enzyme-linked immunosorbent fluorescence assay and high-pressure liquid chromatography for analysis of humoral immune responses to Coxiella burnetii proteins. J Clin Microbiol 26:2520-2525, 1988 61. Blondeau JM, Williams JC, Marrie TJ: The immune response to phase I and phase II Coxiella burnetii antigens as measured by Western immunoblotting. Ann N Y Acad Sci 590:187-202, 1990 62. Willems H, Thiele D, Glas-Adollah-Baik Kashi M, et al: Immunoblot technique for Q fever. Eur J Epidemiol 8:103-107, 1992 63. Peacock MG, Philip RN, Williams JC, et al: Serological evaluation of O fever in humans: Enhanced phase I titers of immunoglobulins G and A are diagnostic for Q fever endocarditis. Infect Immun 41:1089-1098, 1983 64. Tissot-Dupont H, Thirion X, Raoult D : Q fever serology: Cutoff determination for microimmunofluorescence. Clin Diagn Lab Immunol 1:189-196, 1994 65. Kovacova E, Kazar J, Spanelova D: Analysis of antibody response in humans and goats with the use of different Coxiella burnetii antigenic preparations, in Kazar J, Toman R (eds): Rickettsiae and Rickettsial Diseases. Bratislava, Slovakia, Slovak Academy of Sciences, 1996, pp 463-468 66. Peter O, Flepp M, Bestetti G, et al: Q fever endocarditis: Diagnostic approaches and monitoring of therapeutic effects. Clin Invest 70:932-937, 1992 67. Guigno D, Coupland B, Smith EG, et al: Primary humoral antibody response to Coxiella burnetii, the causative agent of Q Fever. J Clin Microbiol 30:1958-1967, 1992 68. McDade JE: Diagnosis of rickettsial diseases. A perspective. Eur J Epidemiol 7:270-275, 1991 69. Marrie TJ, Pollak PT: Seroepidemiology of Q fever in Nova Scotia: Evidence for age dependent cohorts and geographical distribution. Eur J Epidemiol 11:47-54, 1995 70. Fournier PE, Casalta JP, Habib G, et al : Modification of the diagnostic criteria proposed by the Duke endocarditis service to permit improved diagnosis of Q fever endocarditis. Am J Med 100:629-633, 1996 71. Dupuis G, Peter O, Peacock M, et al: Immunoglobulin responses in acute Q fever. J Clin Microbiol 22:484-487, 1985 72. Raoult D: Treatment of Q fever. Antimicrob Agents Chemother 37:1733-1736, 1993
(for query) fever is a worldwide zoonosis. The disease was described first in 1937 by Edward Holbrook Derrick (1898-1976), a public health official in Queensland, Australia, while investigating an outbreak of an unknown febrile illness among abattoir workers. Sir Frank McFarlane Burnet and Mavis Freeman isolated a gram-negative intracellular bacterium from a mouse infected with material sent from Derrick. At the same time, Davis and Herald Rae Cox at the Rocky Mountain Laboratory in Montana isolated an organism from ticks collected near Nine Mile Creek. In 1938, researchers found that the Nine Mile agent and the Q fever agent were identical. The agent was named Coxiella burnetii to honor Cox and Burnet. Since then, C burnetii has been isolated from several animal species. Farm animals and pets are the main reservoirs of infection for humans. Humans acquire the infection mainly via inhalation of infected aerosol particles or ingestion of unpasteurized dairy products. Q fever manifests with a wide spectrum of clinical syndromes. After acquiring primary infection with C burnetii, one-half of patients remain asymptomatic. Among those who are symptomatic, acute Q fever most frequently manifests as a self-limited febrile illness, pneumonia, or hepatitis. Endocarditis is the predominant
Q
From the Unite´ des Rickettsies, CNRS UMR 6020, Faculte´ de Me´decine de Marseille, Marseille, France. Address correspondence to Bernard La Scola, MD, PhD, Unite´ des Rickettsies, CNRS UMR 6020, Faculte´ de Me´decine de Marseille, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France; e-mail: [email protected] Copyright 2002, Elsevier Science (USA). All rights reserved. 1045-1870/02/1304-0006$35.00/0 doi:10.1053/spid.2002.127199
form of chronic Q fever. Despite the availability of wellestablished epidemiologic associations and reliable serologic tests, Q fever infrequently is diagnosed in the clinical setting.1 The diagnosis of Q fever relies mainly upon serology, the method most commonly used being the immunofluorescence assay. This serologic testing always should be done in a patient with a febrile illness and blood cultures that yield negative results.
Q Fever The Bacterium Coxiella burnetii is a small, obligate intracellular, gram-negative bacterium. It is the single species of the genus Coxiella, which belongs to the gamma subdivision of Proteobacteria. Based on 16S rRNA sequence analysis, C burnetii is phylogenetically closest to Legionella spp. C burnetii demonstrates marked resistance to physicochemical agents because of its spore-like form. It resists a temperature of 60°C for 60 minutes and exposure to 0.5 percent formalin for 4 days, and it can survive in soil and milk for several months. In cell cultures or embryonated eggs, C burnetii exhibits phase variation linked to a chromosomal deletion that leads to a lipopolysaccharide shift, with the highly virulent phase I form shifting to the avirulent phase II form. Isolates from different areas of the world exhibit a low degree of genetic heterogeneity.1,2
Epidemiology Q fever has been described in every country except New Zealand. Most cases have been reported from Europe, mainly France, England, Switzerland, Germany, Spain, Greece, Bulgaria, and Israel. Nova Scotia, Canada accounts
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for most of the cases reported from North America. Farm animals, namely cattle, sheep, and goats, are the main reservoirs of C burnetii infection for humans. High numbers of the bacterium are found in the placenta of infected parturient animals and are shed into the environment after labor or abortion. Transmission of infection to humans is accomplished mainly via inhalation of contaminated aerosols generated in this way. Spread of infected aerosols by wind has been reported and may explain cases with no apparent contact with sources of infection.1 In Europe, most cases of Q fever occur during the first 6 months of the year as a result of the heavily contaminated environment after lambing season. Occasional contact with only a small number of bacteria may be sufficient to cause illness.1,3 Outbreaks of Q fever after contact with the birth products of cats, dogs, and other pets also have been reported, mainly from North America, and show no seasonality.4,5 C burnetii also has been isolated from birds and domestic poultry, and humans4,5 may be infected via inhalation of infected fomites.6 The incubation period of acute Q fever ranges from 1 to 3 weeks among patients infected via the respiratory route and depends on the infectious burden.1 Ingestion of unpasteurized dairy products constitutes another source of infection with C burnetii. Cases of human Q fever acquired after contact with parturient products of an infected woman (in an obstetrician), through transplacental transmission during human pregnancy, and through blood and sexual transmission, also have been reported.1
Clinical Manifestations The incidence of Q fever pneumonia is difficult to estimate because of the absence of systematic investigations for infections of C burnetii worldwide. In Nova Scotia, Canada, an active surveillance of the role of Q fever in communityacquired pneumonia is ongoing. Among 149 patients with atypical pneumonia, C burnetii was the third most frequently identified agent (2.7% of the cases), after Mycoplasma pneumoniae and Chlamydia pneumoniae (22.8% and 10.7%, respectively).1 Q fever pneumonia in children has been reported rarely in the literature and most probably remains underdiagnosed.7,8 Patients with Q fever pneumonia present with high-grade fever, in association with severe headache and constitutional symptoms, such as fatigue and myalgia. The incidence of cough ranges from 24 to 90 percent and is productive in one-third of them. Respiratory distress and thoracic pain also are encountered frequently. On auscultation, inspiratory crackles predominate. The most common chest radiographic patterns are single or multiple, frequently bilateral infiltrates, and reticular markings, with a predilection for the lower lobes. Atelectasis and pleural effusion also are described frequently. The course of Q fever pneumonia usually is mild to moderate, compatible with “atypical pneumonia.” However, rapid progression to respiratory failure has been reported.1,7-9 A fatal Q fever pneumonia in an 11-year-old boy with chronic granulomatous disease has been described.10 No specific treatment against Q fever was administered in that case. C burnetii was identified in autopsy lung tissue.
Pathogenesis and Immunity After primary infection has occurred, C burnetii enters monocytes and macrophages via attachment to integrins. Depending on the route of infection (respiratory or gastrointestinal), alveolar macrophages in the lungs or Kupffer cells in the liver are the primary target cells. The avirulent phase II form is destroyed rapidly via the phagolysosomal pathway, whereas the virulent phase I form remains viable. Within macrophages, C burnetii exists in large acidic vacuoles that promote its metabolism and multiplication.1,2 During acute infection, IgM antibodies against phase I and II C burnetii antigens and IgG antibodies against phase II antigens develop. In patients with chronic Q fever, high levels of IgG and IgA antibodies against phase I and II are present. Control of C burnetii infection depends on T cell– mediated immune mechanisms. However, complete eradication of bacteria is not achieved. Patients with malignancies, those infected with human immunodeficiency virus (HIV), and pregnant women may experience relapse of infection, after having an initial response, that may become chronic and frequently fatal. Dysregulation of cytokine responses appears to explain the impaired immunity in such patients. Biopsy specimens taken during acute infection with C burnetii reveal a profound cellular response with a few organisms. However, during chronic infection, biopsy specimens demonstrate high numbers of organisms but a limited cellular response.1,2
Laboratory Diagnosis Nonspecific Laboratory Diagnosis In patients with acute Q fever,11 the leukocyte count usually is normal. However, 25 percent of patients have an elevated white blood cell count (WBC) that ranges from 14 to 21 ⫻ 109/L. The erythrocyte sedimentation rate may be elevated. Thrombocytopenia is noted in 25 percent of cases. Liver enzymes are elevated in as many as 85 percent of cases. The increase in transaminase rate usually is moderate. During an episode of prolonged fever, the association of a normal WBC count, thrombocytopenia, and elevated hepatic enzymes are diagnostic signs of Q fever. In Q fever meningoencephalitis, a mild lymphocytic pleiocytosis frequently is noted in the spinal fluid. Autoantibodies are found frequently during the course of Q fever, but their real significance remains unknown.12-15 In chronic Q fever, clinical and biological symptoms are related to the predominantly cell-mediated inflammatory response to the microorganism.11,16,17 Conventional blood cultures remain negative. The laboratory manifestations of an inflammatory syndrome are the usual ones and include anemia, elevated erythrocyte sedimentation rate, and polyclonal hypergammaglobulinemia. The WBC count may be normal, elevated, or decreased. Thrombocytopenia and elevated hepatic enzyme levels are found commonly. Renal involvement is a common event and is characterized by elevated creatinine levels and microhematuria. Monoclonal immunoglobulins rarely are observed,18 whereas cryoglobulins are found fre-
Current Laboratory Diagnosis of Q Fever quently. Autoantibodies also are frequent findings in chronic Q fever.12,13
Specific Laboratory Diagnosis Samples. C burnetii is a very infectious disease. Thus, only biosafety level 3 laboratories and experienced personnel should be allowed to manipulate contaminated specimens and cultivate this microorganism from clinical samples. Several human specimens are suitable for the detection of C burnetii, but their availability depends on the clinical presentation. DNA amplification may be performed on blood, cerebrospinal fluid, bone marrow, cardiac valve biopsy, vascular aneurysm or graft, bone biopsy, liver biopsy, milk, placenta, fetal specimens in case of an abortion, and cell culture supernatants. Blood should be collected on EDTA or sodium citrate, and the leukocyte layer should be saved for gene amplification. Solid specimens should be kept frozen at -80°C before testing. Cultivation of C burnetii may be obtained from the buffy coat of heparinized blood, whole blood, plasma, bone marrow, cerebrospinal fluid, cardiac valve biopsy, vascular aneurysm or graft, bone biopsy, liver biopsy, milk, placenta, and fetal specimens in case of an abortion, but not from blood collected on EDTA or sodium citrate. All specimens, excluding whole blood, should be stored at -80°C and be forwarded on dry ice to the diagnostic laboratory. Whole blood should be kept at 4°C. Furthermore, red blood cells should not be inoculated onto shell vials because they lead to a high background on examination with ultraviolet light. Pathology, Immunohistochemistry. The immune response during Q fever is associated with an inflammatory reaction that results in formation of granulomatous lesions most commonly involving the lungs, liver, and bone marrow. The histology of Q fever pneumonia in humans rarely has been studied.19-22 Interstitial edema and infiltration by lymphocytes and macrophages occur. Alveolar spaces are filled with histiocytes, and intra-alveolar focal necrosis and hemorrhage have been described. A necrotizing bronchitis and bronchiolitis may be encountered. Microorganisms usually are lacking in these lesions. The hepatic lesions are different in acute and chronic Q fever. In acute cases, the characteristic findings are granulomatous lesions containing the so-called doughnut granuloma, which consists of dense fibrin rings surrounding a central lipid vacuole.23-27 Granulomatous changes and necrosis have been noted in bone marrow.27-29 In chronic cases, pathologic findings are nonspecific with lymphocytic infiltration and foci of spotty necrosis.19 The vegetations in Q fever endocarditis often are smooth and nodular. The valve often is infiltrated with foamy macrophages that are filled with C burnetii cells. The detection of C burnetii in tissues can be achieved on fresh or formalin-fixed and paraffin-embedded samples. Valvular or vascular samples are most valuable. Several techniques are available and include immunoperoxidase,30 a capture system [enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunofluorescent assay (ELIFA)31], or monoclonal antibodies.31-33 The last technique also can be employed for antigenic detection in paraffin-embedded tissues.34
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DNA Detection. The polymerase chain reaction (PCR) has been used successfully to detect C burnetii DNA in cell cultures and clinical samples.35 The availability of primers derived from genes specific to C burnetii has allowed a simple and reliable method for the detection of this bacterium, even in paraffin-embedded tissues.35,36 Moreover, PCR has proven to be more sensitive for retrospective diagnosis on frozen samples and for follow-up of patients treated for chronic Q fever than are standard culture techniques.35 In our experience, specimens kept frozen at -80°C are suitable for PCR for several years. In our laboratory, we routinely use primers derived from the htpAB-associated repetitive element.37,38 This element exists in at least 19 copies in the C burnetii Nine Mile I genome, and PCR based upon this gene is very sensitive.38 Culture. The isolation of C burnetii must be carried out only in biosafety level 3 laboratories because of its extreme infectivity. This microorganism can be isolated by inoculating specimens onto conventional cell cultures (monkey kidney cells—Vero cells), into embryonated hen yolk sacs,39 or into laboratory animals, such as mice or guinea pigs.40 Embryonated eggs die 7 to 9 days after inoculation. Guinea pigs develop fever 5 to 8 days after intraperitoneal inoculation. The spleen is the most valuable organ for the recovery of C burnetii. Ground spleen extracts should be inoculated subsequently into embryonated eggs. The recent development of a cell microculture system from a commercially available method for virus culture, the shell vial cell culture system, has allowed for improvement in the isolation of intracellular bacteria, especially C burnetii.41,42 Specimens are inoculated onto human embryonic lung fibroblasts grown on a 1-cm2 coverslip within a shell vial. A 1-hour centrifugation step enhances attachment and penetration of bacteria into cells. After an incubation period of 6 days, detection of C burnetii within the cells is achieved by microscopic examination after staining. The organism appears as short rods that are not Gram-stained but are visible after Giemsa or Gimenez staining.43 The identification of C burnetii within the cells is performed with a direct immunofluorescence assay using polyclonal or monoclonal anti–C burnetii antibodies conjugated to fluorescein isothiocyanate.42 The detection by antibodies may be confirmed by PCR performed on the shell vial supernatants. Best results for culture are obtained if clinical specimens are collected before initiating antibiotic therapy.44 Fifteen percent of untreated patients with Q fever pneumonia have blood cultures that test positive by this method, as do 53 percent of endocarditis cases.44,45 Serologic diagnosis. Because arriving at the clinical diagnosis is difficult, in most instances, the diagnosis of Q fever relies upon serology. Several methods have been described: microagglutination,46-48 complement fixation,49-51 radioimmunoassay,52 indirect immunofluorescence antibody tests (immunofluorescence assay),51,53 indirect hemolysis test,54 ELISA,55-59 ELIFA,60 dot immunoblotting, and Western immunoblotting.61,62 Criteria to be taken into account in choosing a diagnostic test include its specificity, sensitivity, positive predictive value, cost, and the amount of antigen required. The most reliable and commonly used
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methods are indirect immunofluorescence, complement fixation, ELISA, and microagglutination. Only the first 2 are commercially available and will be described in this article. Currently, the immunofluorescence assay is the reference method for the serodiagnosis of Q fever.63 It is the simplest and one of the most accurate serologic techniques. To prepare antigens for this test, phase II C burnetii Nine Mile reference strain is grown in confluent layers of L929 mouse fibroblasts, and phase I antigens are obtained from the mice spleens inoculated with phase II organisms.64 This method of preparation has been demonstrated to yield antigens with the highest sensitivity for C burnetii antibody detection.65 In our laboratory, we use a micro-immunofluorescence technique, which requires very small amounts of antigens. Sera are diluted in phosphate buffered saline with 3 percent nonfat powdered milk to avoid nonspecific fixation of antibodies. This method can be used to determine antibodies to phases I and II in the IgG, IgM, and IgA fractions. However, test results can be confounded by the presence of a rheumatoid factor. Thus, a rheumatoid factor absorbant is used to remove IgG before the determination of IgM and IgA.64 The choice of a negative cutoff titer depends upon the source and purity of the antigen and the amount of background antigen stimulation in the population to be studied. We use a 1:50 dilution as our first positive dilution.64 Screening is performed with anti–phase II anti-immunoglobulins with a 1:50 dilution for the tested sera. Positive sera then are diluted serially and tested for the presence of anti–phase I and II IgG, IgM, and IgA. Seroconversion usually is detected 7 to 15 days after the onset of clinical symptoms. Approximately 90 percent of patients have detectable antibodies by the third week. Complement fixation is very specific, although less so than is immunofluorescence assay, but it lacks sensitivity.51 Sera are heat-inactivated before testing against phase II antigens.49 This method detects both anti–phase I and II antibodies. However, a prozone phenomenon may be present with serum specimens from patients with chronic Q fever that could result in a false-negative test. Complement fixation also is more time-consuming than is immunofluorescence assay.66 Moreover, cross-reaction with hen egg antigens may result in false-positive results. The interpretation of results requires acute and convalescent phase serum samples. Seroconversion is detected later with the complement fixation test than with immunofluorescence assay or ELISA (between 10 and 20 days after the onset of symptoms).51,67 The predictive value of positive and negative results is affected by the prevalence of the disease, which creates a certain degree of background antigen stimulation in the studied population.68 Therefore, the determination of cutoff values is very important for the interpretation of the serologic results. For example, we use a 1:50 serum dilution for immunofluorescence assay screening, whereas Marrie and Pollak69 used a 1:8 dilution. Whereas a diagnostic test must be very sensitive, a seroepidemiology test must be very specific to prevent false-positive results caused by cross-reacting antibodies. The antigenic variation of C burnetii is extremely useful in differentiating acute and
chronic illness. In acute Q fever, antibodies to phase II antigens predominate, and their titer is higher than the phase I antibody titer. As with many other infectious diseases, IgM antibodies are the first to appear. On the other hand, in chronic forms of the disease, such as endocarditis, elevated anti–phase I antibodies are detected uniformly. For cutoff values in the immunofluorescence assay test, Tissot-Dupont et al64 recommend titers of anti–phase II IgG of 200 or greater and titers of anti–phase II IgM of 50 or greater for the diagnosis of acute Q fever, and titers of anti–phase I IgG of 800 or greater for the diagnosis of chronic Q fever. They also have demonstrated that anti– phase I IgA titers did not contribute to the diagnosis of chronic Q fever. Recently, we demonstrated that an anti– phase I IgG C burnetii titer of 1:800 or greater was diagnostic of Q fever in a case of endocarditis, and, thus, we have modified the Duke criteria for the diagnosis of Q fever endocarditis to include 1 positive blood culture for C burnetii and a phase I IgG titer of 1:800 or greater as major criteria for the diagnosis of Q fever endocarditis.70 A complement fixation titer of 1:40 is diagnostic for acute Q fever,67 and a 1:200 titer of antibody to phase I is diagnostic for chronic Q fever.66 In acute Q fever, immunofluorescence assay titers reach their maximum levels 4 to 8 weeks after the onset of disease and then decrease gradually during the ensuing 12 months.67 Dupuis et al71 have shown that IgM titers declined to undetectable levels after 10 to 12 weeks, whereas the decline took 17 weeks in the study of Field et al.53 Complement fixation and immunofluorescence assay can be combined for the follow-up. A drop in complement fixation titer often implies resolution and will occur before an immunofluorescence assay drop. The persistence of high levels of anti–phase I antibodies despite appropriate treatment, or the reappearance of such antibodies, should raise suspicion of possible chronic Q fever. Patients with valvular or vascular abnormalities, those who are immunodeficient, and pregnant women should have repeated C burnetii serology tests if they have a medical history of acute Q fever or a prolonged and unexplained febrile episode. In the case of an acute Q fever in such a patient, an immunofluorescence assay test should be done monthly for at least 6 months. The follow-up of patients treated for chronic Q fever also should be done serologically. During therapy, serologic testing should be performed once monthly for 6 months and every 3 months thereafter. The levels of antibodies decrease very slowly. When present, IgM antibodies disappear first, then IgA antibodies, but IgG titers remain positive for years. Antimicrobial treatment can be stopped after 18 months to 3 years if the anti–phase I IgG titer by immunofluorescence assay is below 1:400 and anti–phase I IgA is undetectable.14,72
Conclusion Q fever remains a poorly understood disease despite recent advances in the knowledge of its immunopathology. Acute cases present most often as asymptomatic infections, in-
Current Laboratory Diagnosis of Q Fever cluding flulike syndrome, pneumonia, or hepatitis. Host factors probably play an important role in the development of chronic disease, which presents in most cases as a blood culture–negative endocarditis. Although its exact prevalence is unknown, the number of cases of Q fever most likely is underestimated. Therefore, such a diagnosis must be considered in cases of unexplained fever, especially if the fever occurred after contact with possibly contaminated mammals. The best tests for diagnosis are those that permit the direct detection of bacteria. They include the shell vial cell culture, PCR, and the immunodetection on tissue biopsies. All these techniques require a level 3 biosafety laboratory and trained personnel because of the extreme infectivity of C burnetii. In chronic cases, the techniques that allow direct detection of C burnetii in blood or tissues should be used before the beginning of therapy. As for indirect specific diagnosis, the technique to be used should be very sensitive and should detect antibodies early in the course of the disease. Although many techniques have been described, immunofluorescence assay is the reference method. It is both very specific and sensitive. In the case of acute Q fever, diagnosis can be confirmed by an immunofluorescence assay titer greater than the cutoff value (which has to be determined for each area) or by a 4-fold increase in the antibody titer detected by immunofluorescence assay, complement fixation, ELISA, or microagglutination.
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